Nitriles are exceedingly versatile compounds that can be used in the synthesis of a wide variety of compounds, including amines, amides, amidines, carboxylic acids, esters, aldehydes, ketones, imines, and heterocyclics. One of the most important commercially important nitriles is acetonitrile which is a common solvent. Other nitrile compounds are used as herbicides or in the synthesis of detergents or antiseptics. Another of the most commercially important nitriles is acrylonitrile, which is used to make acrylamide, acrylic acid, acrylic fibers, copolymer resins and nitrile rubbers.
One method of production of acrylonitrile is by use of the SOHIO/BP process, which entails the direct ammoxidation of propene (a/k/a propylene) by ammonia vapors in air in the presence of a catalyst (see generally, Acrylonitrile, 1979, Process Economics Program Report, Stanford Research International, Menlo Park, Calif.; Weissermel and Arpe, 1978, Industrial Organic Chemistry, Verlag Chemie--Weinheim N.Y., pp. 266-270). The waste stream from this process contains a complex mixture of nitriles, including dinitriles, amides and acids at high concentrations. More particularly, the wastestream generally contains nitriles such as acetonitrile, acrylonitrile, succinonitrile and fumaronitrile as well as acrylamide. In addition, cyanide(s) at variable and/or high concentrations is/are often present. The wastestream generally contains high and/or variable concentrations of ammonium sulfate. This hazardous waste effluent cannot be released into the environment due to its toxicity and in the United States is usually disposed of by deep well injection into sub-surface formations. Such disposal cannot be considered to be a "treatment" of the wastestream, but rather is analogous to the process of landfilling.
Outside the United States, it has been common practice to "treat" the wastestream from the production of acrylonitrile by diluting the wastestream to a low total nitrile concentration of about 250 ppm or less and treating by conventional aerated biological wastestream systems, i.e., activated sewage sludge, after wet/air oxidation, which removes volatiles and partially oxidizes many of the organic constituents. Such method of "treatment" is not suitable to efficient disposal of a nitrile production facility wastestream for the following reasons: (1) wet/air oxidation causes volatile compounds to be stripped, creating an air emission problem; (2) dilution of the wastestream necessitates large wastewater treatment facilities to handle the flow and long residence time required to obtain adequate "treatment"; and (3) combination of wet/air oxidation with biological treatment by activated sludge results in high treatment costs. There remains a long, deep felt need in the nitrile production industry for an efficient, cost effective, environmentally sound method to dispose of the effluent of nitrile production plants.
It has long been known that certain microorganisms are useful to convert a nitrile compound to its corresponding amide or acid compound biologically. Both the scientific and patent literature contain numerous references describing the use of nitrile converting microorganisms for the production of specialty chemicals, for example, acrylamide and acrylic acid or acrylate from acrylonitrile. See generally, Kobayshi et al., 1992, Trends Biotechnol. 10:402-408. The nitrile converting microorganisms have been shown to have activities including, nitrilase, which converts a nitrile compound to its corresponding acid compound; nitrile hydratase, which converts a nitrile compound to its corresponding amide compound; and amidase, which converts an amide compound to its corresponding acid compound.
To the knowledge of the present inventor, however, in all these specialty chemical productions using nitrile degrading microorganisms, only a single compound has been employed to induce the relevant activity and only a single nitrile compound has been converted to produce a single desired specialty compound.
2.1. Microorganisms which can utilize a Nitrile Compound
The literature contains certain references which disclose a number of microorganisms which can utilize a nitrile or an amide compound as the sole source of carbon and/or nitrogen.
For example, Asano et al., 1982, Agric. Biol. Chem. 46:1165-1174, describe an isolated strain of Arthrobacter which is able to grow using acetonitrile as a sole source of carbon and nitrogen.
Nawaz et al., 1989, 43rd Purdue Ind. Waste Conf. Proc., pp. 251-256 (Nawaz, 1989), describe the isolation of a Pseudomonas aeruginosa strain which is able to utilize various nitrile compounds, including acetonitrile, as a sole source of carbon and energy. However, this strain is unable to utilize other nitrile compounds such as acrylonitrile, acrylamide, benzonitrile and malononitrile.
Nawaz et al., 1992, Appl. Environ. Microbiol. 58:27-31 (Nawaz), describe a Klebsiella pneumonia NCTR1 strain which, after acclimation using benzonitrile, could degrade a mixture of benzonitrile and one other nitrile selected from butyronitrile, acetonitrile, glutaronitrile, propionitrile, succinonitrile and methacrylonitrile. In complete contrast to the present method for induction which does not require the presence of an aromatic nitrile, Nawaz's microorganism required benzonitrile in order to induce the ability to degrade the mixtures of benzonitrile and one other nitrile. Moreover, and most importantly, in order to achieve degradation of any of the mixtures of nitriles, benzonitrile had to be present. Since the nitrile wastestream of a nitrile production facility does not contain benzonitrile, this organism and the method disclosed by Nawaz would be completely impractical and, in fact, inoperative for treating such wastestream.
Chapatwala et al., 1993, App. Biochem. Biotech. 39/40:655-666, describe the isolation of a Pseudomonas putida strain which is capable of utilizing acetonitrile as a sole source of carbon and nitrogen. However, there is no disclosure of utilization of any other nitrile-containing compound by the strain.
Narayanasamy et al., 1990, Indian J. Exp. Biol. 28:968-971, disclose the utilization of acrylonitrile, acetonitrile, acrylamide and acetamide by an Arthrobacter sp. individually. There is no indication that the bacterial strain is able to degrade dinitriles or a mixture of nitriles.
O'Grady and Pembroke, 1994, Biotech. Letters 16:47-50, describe the isolation of an Agrobacterium sp. and the ability of the isolated strain to utilize or break down a number of different nitrile compounds individually. There is no indication that the isolated strain would be able to utilize or break down a mixture of the nitrile compounds.
Martinkova et al., 1992, Folia Microbiol. 37:373-376, disclose the isolation of several bacterial strains, including Corynebacterium sp. strain 3 B and Agrobacterium radiobacter strain 8/4/1, which are able to utilize acetonitrile as a sole source of carbon and nitrogen. There 30 is no disclosure that the strains are able to utilize any other nitrile compound or a mixture of nitrile compounds.
Nawaz et al., 1994, Appl. Environ. Microbiol. 60:3343-3348, describe the isolation of a bacterium, tentatively identified as a Rhodococcus sp., from soil contaminated with the herbicide alachlor. This bacterium was shown to be able to use acrylonitrile as a sole source of carbon and nitrogen.
Nawaz et al., 1993, Can. J. Microbiol. 39:207-212, describe the isolation of a Pseudomonas sp. and Xanthomonas maltophilia, which each can utilize acrylamide as a sole source of carbon and nitrogen.
Armitage et al., International Patent Publication WO 97/06248, published Feb. 20, 1997, discloses methods for producing an amidase by culturing a suitable microorganism in the presence of an amide or an amide precursor, such as a nitrile, or a mixture thereof, under continuous culture, carbon-limiting conditions in which the amide or amide precursor forms at least 20% mol and preferably substantially all of the carbon. Suitable microorganisms include Pseudomonas, Rhodococcus, etc. Also disclosed are methods for producing a nitrilase by culturing a microorganism in the presence of a nitrile or a nitrile precursor, or a mixture thereof, under continuous culture carbon-limiting culture conditions. Suitable microorganisms include Nocardia, Rhodococcus spp., including Rhodococcus ATCC 39484, etc. The induced enzyme is then used, inter alia, to convert a nitrile or amide to its corresponding acid.
Although microorganisms which utilize a nitrile compound might possibly be useful to remove a single nitrile compound from a nitrile containing composition, the use of such organisms to aid in the disposal of the wastestream of a nitrile production facility is not to be expected because nitrile utilization is dependent upon the expression of a nitrilase or nitrile hydratase specific to the single nitrile compound utilized. Expression of a specific nitrilase or nitrile hydratase does not assure that the microorganism will have the ability to convert the mixture of nitrile compounds or the mixture of nitrile and amide compounds present in the high concentrations found in the wastestream of a nitrile production facility.
2.2. Treatment of Nitrile Wastes Including a Wastestream of a Nitrile Production Plant
A number of references describe attempts to provide a microbiological method to remove a nitrile from a nitrile waste, including the wastestream of a nitrile production plant.
U.S. Pat. No. 3,940,332 to Kato et al. (Kato) describes the use of an isolated bacterial strain, Nocardia rubropertincta, ATCC Accession No. 21930, in combination with activated sludge from a sewage treatment plant to degrade a wastestream containing nitriles and inorganic cyanides. Kato also indicates that the bacterial strain is able to degrade nitriles, including, acetonitrile, acrylonitrile, propionitrile, butylonitrile, crotononitrile, fumaronitrile, valeronitrile, glutaronitrile, and benzonitrile, although no indication is given of the amount of each of such nitriles or conditions under which the nitriles are degraded or whether the nitriles can be degraded together. There is no indication that the strain disclosed by Kato can degrade or detoxify a mixture of nitrile compounds. Further, the nitrile waste treated by Kato was a low strength waste, 50-250 ppm total nitrile concentration. Moreover, the present inventor has tested the strain disclosed by Kato and found that the strain does not remove acetonitrile from a mixture of nitrites with the same efficiency as can be accomplished using the methods of the present invention.
Sunarko and Meyer, 1989, DECHMA Biotech. Conf. 3:859-862, disclose that lyophilized cells of Mycobacterium UBT5, Bacillus UBT2, Corynebacterium UBT9, and Flexibacter UBT4, which had been induced by growing the cells in the presence of 2-pentenenitrile, were able to degrade small quantities of acetonitrile found in laboratory HPLC column effluent.
Brown et al., 1980, Water Res. 14:775-778, disclose that acrylamide spiked at concentrations of 0.5 ppm to 5 ppm into natural and polluted waters of the environment resulted in the degradation of the acrylamide.
Kincannon et al., 1983, Journal WPCF 55:157-163, disclose that a mixture of microorganisms isolated from a municipal activated sludge water treatment plant was able to degrade acrylonitrile after a one month acclimation period. Further, the authors also showed that acrolein could be similarly degraded by the mixture of microorganisms.
Donberg et al., 1992, Environ. Toxicol. Chem. 11:1583-1594, disclose that a mixture of microorganisms found in soil was able to degrade acrylonitrile under aerobic conditions. Some mixtures were able to degrade 10-100 ppm acrylonitrile on the order of 2 days. However, at higher concentrations of acrylonitrile (1000 ppm), degradation was inhibited. The authors speculated that the inhibition was due to inhibitory effects of the parent acrylonitrile compound.
Knowles and Wyatt, European Patent No. 274 856 B1 and Wyatt and Knowles, 1995, Biodegradation 6:93-107, describe the degradation of a mixture of nitrile and amide compounds from the wastestreams of a nitrile production plant (using the BP/SOHIO process of acrylonitrile production) by a mixture of microorganisms. The use of a mixture of microorganisms rather than a pure culture, is a serious drawback of the method of Knowles and Wyatt. It is difficult to maintain a mixed culture, for as the conditions of the reaction change, certain strains within the mixed culture will be favored at different times for growth such that the efficacy of degradation can be decreased.
There are a number of disadvantages associated with the above references. For example, many of the individual microbial strains described above have only a limited range of nitrile or amide compounds which they can degrade. The time required for degradation/utilization of nitrile and amide compounds is on the order of days or weeks. Further, disposal by traditional activated sludge treatment has its own drawbacks, such as the large amount of biomass produced, which must eventually be disposed. Moreover, and most importantly, the use of a mixture of microorganisms rather than pure cultures, makes it very difficult because it is difficult to maintain the mixed culture. As conditions of the reaction change, certain strains within the mixed culture will be favored at different times for growth, such that over time the characteristics of the mixed culture will change and the efficacy of degradation can decrease. The mixed culture is not easily reproduced or maintained.
2.3. Treatment of Polyacrylamide Preparations to Remove the Acrylamide Monomer
A number of references describe attempts to provide a microbiological method to remove an unwanted acrylamide monomer from a polyacrylamide preparation.
Polyacrylamide polymers are widely used in several industries, including sewage treatment, paper manufacturing, mining, and in biological and chemical research. However, their utility is restricted and they cannot be used in connection with foodstuffs because they are generally contaminated with unreacted acrylamide monomer which is a cumulative neurotoxin and a carcinogen. One method of reducing the amount of unreacted monomer is the "heat treatment" method. However, this treatment increases the cost of manufacture and decreases the efficiency of polymer production due to undesired branching of the polymers.
Another method is the use of amidase obtained from a microorganism. For example, Carver et al., European Patent Publications EP 272,025 A2 and EP 2072,026 A2, describe the decomposition of acrylamide in polyacrylamide preparations using an amidase obtained from an organism such as Methylophilis sp. which has been heated to a temperature in the range of 40.degree. C. to 80.degree. C. However, the methods disclosed by Carver et al. do not allow for the removal of acrylamide monomer from a cationic polyacylamide preparation at its native pH value, i.e., a native pH of about 4. See FIG. 6 in EP 272,025 A2, which clearly shows that in order for the acrylamide monomer to be removed from the cationic polymer, the pH must be raised from a pH of 4 to a pH of 6.
U.S. Pat. Nos. 4,687,807 and 4,742,114 to Westgrove et al. describe the production of water-in-oil emulsions of amidase for use in removing unwanted acrylamide monomer from a preformed acrylamide polymer.
Farrar et al., European Patent Publication EP 329,325 A2, describes the removal of acrylamide monomer from polyacrylamide using an aqueous gel of amidase obtained from an amidase-expressing microorganism. Farrar, International Patent Publication WO 92/05205, describes reduction of residual (meth)acrylamide monomer in a polymerized (meth)acrylamide by incorporating amidase into a polymerizable mixture for exothermic polymerization of polymeric (meth)acrylamide.
Armitage et al., International Patent Publication WO 97/06248, published Feb. 20, 1997, also discloses methods for the conversion of meth(acrylamide) to ammonium meth(acrylate) in or after the ploymerization of the acrylamide using an amidase which has been induced in a suitable microorganism obtained by culturing the microorganism in the presence of an amide or an amide precursor, such as a nitrile, or a mixture thereof, under continuous culture, carbon-limiting conditions in which the amide or amide precursor forms at least 20% mol and preferably substantially all of the carbon. Suitable microorganisms include Pseudomonas, Rhodococcus, etc. However, no working examples are provided in which the induced enzyme actually converts the acrylamide monomer in a polyacrylamide preparation, and further, there is no disclosure regarding the pH range in which the conversion reaction could take place.
Citation or identification of any reference in Section 2 or any section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.